CN107778376A - A kind of preparation method and applications of radix tetrastigme polysaccharide - Google Patents
A kind of preparation method and applications of radix tetrastigme polysaccharide Download PDFInfo
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- CN107778376A CN107778376A CN201711200506.7A CN201711200506A CN107778376A CN 107778376 A CN107778376 A CN 107778376A CN 201711200506 A CN201711200506 A CN 201711200506A CN 107778376 A CN107778376 A CN 107778376A
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention belongs to biological technical field, and in particular to the preparation and application of Chinese herbal medicine radix tetrastigme polysaccharide.Using radix tetrastigme cane leaves as raw material, implement successively in accordance with the following steps：（1）Hot water extraction；Collected by suction filtrate；It is concentrated in vacuo；Concentrate adds ethanol；Collect precipitation, i.e. radix tetrastigme Thick many candies.（2）Thick many candies are dissolved in water, add Sevag reagents；Removing protein layer and organic layer are centrifuged after stirring, it is multiple untill albumen is occurred without to repeat this step operation；（3）Polysaccharide after removing protein is dissolved in deionized water, radix tetrastigme polysaccharide one-component is obtained by the ion-exchange chromatographies of DEAE 52 and the sephadex chromatographies of Sephadex G 100.Radix tetrastigme polysaccharide involved in the present invention, body blood pressure and blood lipoid can be significantly reduced, improve antioxidase enzyme activity level in body, and there is certain repair to pancreas islet, there is good medical value and market value.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of preparation and its application of radix tetrastigme polysaccharide.
Background technology
Radix tetrastigme（Tetrastigma hemsleyanum Diels et Gilg）Rectification of name tetratigma hemsleyanum, also known as gold thread
Hoist, hemsley rockvine root, the areas such as dark and damp hillside, mountain valley are grown on, are mainly distributed on China southeast.Southwestern area.Radix tetrastigme is complete
Grass can be used as medicine, and be one of famous Chinese herbal medicine in China, have significant medical value, such as treat viral meningitis, branch gas
Guan Yan, children with high fever induced convulsions, prevention and treating cancer etc..
Diabetes are divided into a patients with type Ⅰ DM and type-II diabetes, wherein a patients with type Ⅰ DM refers to that insulin is relative or definitely divides
Secrete deficiency, it is necessary to rely on insulin and maintain life, type-II diabetes refer to insulin resistance or insensitive, and patient has typical
" three-many-one-little " symptom, i.e., more more drink diuresis of food, weight loss.As living standards of the people are growing, diabetes are suffered from China
Number it is growing day by day.According to statistics, diabetic number in China's is up to ten thousand people more than 9000 at present, occupies the first in the world, two types
Diabetic occupies more than the 80% of diabetes number of patients, and more than 50% type-II diabetes patient has one or more sugar
Urinate disease complication.Diabetic complication includes hypertension, high fat of blood, cardiovascular and cerebrovascular disease etc., and the serious body for damaging the people is good for
Health, therefore it is very urgent to prevent and treat diabetes.
The content of the invention
It is an object of the invention to provide a kind of preparation and its application of radix tetrastigme polysaccharide.
In order to solve the above technical problems, the present invention can be realized by following steps.
The preparation of the radix tetrastigme polysaccharide, comprises the following steps：
（1）The extraction of radix tetrastigme Thick many candies：
Dry radix tetrastigme and boiled after crushed with 80 DEG C of deionized water and carried 3 hours；Filtrate is filtered to obtain, filtrate is dense by 80 degree of water-baths
The a quarter of original volume is reduced to, adds the absolute ethyl alcohol of 5 times of volumes, precipitates left undisturbed overnight；5000 revs/min of centrifugations are received for 10 minutes
Collection precipitation, Thick many candies are obtained after freeze-dried；
（2）Savag method removing proteins.
A1：Sevag reagents are added into the 10wt% radix tetrastigme Thick many candies aqueous solution to be sufficiently stirred 1 ~ 2 hour,；
B1：Then 3500~4500 revs/min centrifuge 10~25 minutes, remove albumin layer and chloroform layer, water layer are repeated to not
Untill there is albumin layer again, obtained aqueous solution is through dialysing and freeze-drying obtains de- albumen radix tetrastigme essence polysaccharide.
（3）The classification of radix tetrastigme essence polysaccharide：
5wt% is taken off into albumen radix tetrastigme polysaccharide to be classified through DEAE- cellulose ion-exchange chromatographies post；3 times of column volumes it is different dense
Sodium chloride solution elution is spent, collects 0.3M sodium chloride eluant components, dialysed through deionized water, be freeze-dried and obtain polysaccharide level
Point.
Step（2）In, Sevag reagents include n-butanol and chloroform；Wherein：The volume ratio of n-butanol and chloroform is：1 ：
4；
Radix tetrastigme polysaccharide component made from the inventive method is accredited as one-component by Sephadex G-100 gel elutions, its
Middle monose composition and molar ratio are respectively Rha：Ara：Man：Glc：:Gal = 006：0.13：0.38：0.09：0.31.Point
Son amount is 9 × 104 ~10.0 × 104Da。
The remarkable advantage of the present invention is：
Polysaccharide is extracted by traditional water extract-alcohol precipitation mode, extraction conditions are simple, and extraction cost is cheap, obtained secondly by two-step purifying
To the polysaccharide of one-component, just with the follow-up related application for carrying out hypoglycemic activity.
Brief description of the drawings
Fig. 1 is the ion-exchange chromatography figure of radix tetrastigme polysaccharide.
Fig. 2 is the Sephadex G-100 gel chromatography figures of radix tetrastigme polysaccharide.
Embodiment
For the further open rather than limitation present invention, below in conjunction with example, the present invention is described in further detail.
Embodiment 1
Implement to prepare successively as follows.
Dry radix tetrastigme powder 500g is weighed, is extracted 3 hours with 2 liters of 80 DEG C of deionized waters；Suction filtration obtains filtrate, at 80 DEG C
The 1/4 of original volume is concentrated under bath temperature；The absolute ethyl alcohol of 5 times of volumes is added, precipitates left undisturbed overnight；5000 revs/min of centrifugations
Precipitation is collected within 10 minutes, 50.45 grams of Thick many candies are obtained after freeze-dried；
50 grams of radix tetrastigme Thick many candies are weighed, are dissolved in 1500 ml deionized waters, add 300 milliliters of Sevag reagents；Chloroform；Just
Butanol=4:1, v/v；It is sufficiently stirred 1 hour, 3500 leave the heart 20 minutes, remove albumin layer and organic layer；Water layer, which is iteratively repeated, to be removed
Albumen step is untill albumin layer is occurred without；Obtained aqueous solution is dialysed 72 hours with deionized water, and albumen three must be taken off by being freeze-dried
27.56 grams of leaf green grass or young crops polysaccharide.
5 grams of de- proteoglycans are dissolved in 50 ml deionized waters, exchanged with 2.6 × 30cmDEAE- cellulose ions
Post separation；Eluted successively with 500 milliliters of water, 0.1M-0.3M sodium chloride solutions, four degree of elution is 8 ml/mins, every 20 milliliters
Eluent collects a pipe, is detected via Phenol sulfuric acid procedure, collects the component that 0.3M sodium chloride elutes, is dialysed with deionized water
After 72 hours, freeze-drying obtains 2.2 grams of radix tetrastigme polysaccharide components.
Radix tetrastigme polysaccharide hypoglycemic is tested.
Take 10 to be only used as blank control group at random from 100 ICR mouse, after remaining mouse fasting 12 hours, press
Alloxan solution is injected intraperitoneally in 100mg/kg dosage.After 72 hours, tail vein blood, mouse blood sugar after measure fasting 12 hours
Value, take diabetic mice of mouse of the blood glucose value more than 11.0mmol/L for shaping.Diabetic mice is divided into 6 groups, often
Group 10, model control group and Normal group gavage give glibenclamide 25mg/ to deionized water, positive controls gavage
Kg, remaining 3 groups are polysaccharide experimental group, give radix tetrastigme polysaccharide dosage 100,200 and 400mg/kg respectively.Daily gastric infusion 1
It is secondary, continuous 35 days, after last dose after all mouse fasting 12 hours, determine blood-sugar content.Experimental result is shown in Table 1.
Influence of the radix tetrastigme polysaccharide of table 1 to blood glucose in diabetic mice.
Note：Compared with model group：*, p ＜ 0.05；*, p ＜ 0.01.
As shown in Table 1, after gastric infusion 35 days, radix tetrastigme polysaccharide and positive controls can significantly drop diabetic mice
Low mouse blood sugar value, wherein polysaccharide 400mg/kg experimental groups can pole significantly reduce mouse blood sugar, effect is said close to glibenclamide
Bright radix tetrastigme polysaccharide can realize its function of blood sugar reduction in the case of oral.
Fig. 1 is the polysaccharide after radix tetrastigme removing protein, is purified by the first round, i.e. knot after DEAE-52 ion-exchange chromatographies
Fruit, as figure shows, co-elute go out 5 components, choose key component and collect progress next step purifying.Fig. 2 is that the second wheel is pure
Change, i.e. the elution profile of sephadex g-100, as shown in Figure 2, only appearance one is unimodal, illustrates the polysaccharide for being purified to this step
It is that molecular weight is homogeneous, the homogeneous one-component polysaccharide of the quantity of electric charge.Application available for hypoglycemic experiment in next step.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
Claims (3)
- A kind of 1. preparation method of radix tetrastigme polysaccharide, it is characterised in that：Comprise the following steps to implement successively：（1）Dry radix tetrastigme powder 500g is weighed, is extracted 3 hours with 2 liters of 80 DEG C of deionized waters；Suction filtration obtains filtrate, at 80 DEG C The 1/4 of original volume is concentrated under bath temperature；The absolute ethyl alcohol of 5 times of volumes is added, precipitates left undisturbed overnight；5000 revs/min of centrifugations Precipitation is collected within 10 minutes, Thick many candies are obtained after freeze-dried；（2）50 grams of radix tetrastigme Thick many candies are weighed, are dissolved in 1500 ml deionized waters, add 300 milliliters of Sevag reagents；Fully Stirring 1 ~ 2 hour, 3500 leave the heart 20 minutes, remove albumin layer and organic layer；Water layer is iteratively repeated removing protein step to not going out Untill existing albumin layer；Obtained aqueous solution is dialysed 72 hours with deionized water, and albumen radix tetrastigme polysaccharide must be taken off by being freeze-dried；（3）5 grams of de- proteoglycans are dissolved in 50 ml deionized waters, with 2.6 × 30cmDEAE- cellulose ion exchange columns Separation；Eluted successively with 500 milliliters of water, 0.1M-0.3M sodium chloride solutions, the ml/min of elution speed 8, every 20 milliliters are washed De- liquid collects a pipe, is detected via Phenol sulfuric acid procedure, collects the component that 0.3M sodium chloride elutes, with deionized water dialysis 72 After hour, freeze-drying obtains radix tetrastigme polysaccharide component.
- A kind of 2. preparation method of radix tetrastigme polysaccharide according to claim 1, it is characterised in that：Sevag reagents are specially： Count by volume, chloroform；N-butanol=4:1.
- 3. application of the radix tetrastigme polysaccharide prepared by method a kind of as claimed in claim 1 on hypoglycemic drug is prepared.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108976310A (en) * | 2018-08-06 | 2018-12-11 | 福建三叶青药业科技开发有限公司 | A kind of preparation method of radix tetrastigme zinc polysaccharide anti-oxidative agent |
| CN109620893A (en) * | 2019-01-08 | 2019-04-16 | 浙江大学 | The purposes of the anti-senility life-prolonging of radix tetrastigme flavones |
| CN109705229A (en) * | 2018-12-14 | 2019-05-03 | 浙江中医药大学 | A kind of radix tetrastigme polysaccharide and its preparing the application in immunomodulator |
| CN110483656A (en) * | 2019-09-10 | 2019-11-22 | 福州大学 | A kind of preparation of radix tetrastigme polysaccharide antibacterial bacteriostatic mouthwash |
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| CN105504076A (en) * | 2015-12-01 | 2016-04-20 | 浙江中医药大学 | Tetrastigma hemsleyanum Diels et Gilg root tuber polysaccharide with anti-pyretic and anti-inflammatory functions and application of Tetrastigma hemsleyanum Diels et Gilg root tuber polysaccharide |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108976310A (en) * | 2018-08-06 | 2018-12-11 | 福建三叶青药业科技开发有限公司 | A kind of preparation method of radix tetrastigme zinc polysaccharide anti-oxidative agent |
| CN109705229A (en) * | 2018-12-14 | 2019-05-03 | 浙江中医药大学 | A kind of radix tetrastigme polysaccharide and its preparing the application in immunomodulator |
| CN109705229B (en) * | 2018-12-14 | 2021-06-04 | 浙江中医药大学 | Radix tetrastigme polysaccharide and application thereof in preparing immunomodulator |
| CN109620893A (en) * | 2019-01-08 | 2019-04-16 | 浙江大学 | The purposes of the anti-senility life-prolonging of radix tetrastigme flavones |
| CN110483656A (en) * | 2019-09-10 | 2019-11-22 | 福州大学 | A kind of preparation of radix tetrastigme polysaccharide antibacterial bacteriostatic mouthwash |
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